Taxonomic Positions and Secondary Metabolite-Biosynthetic Gene Clusters of Akazaoxime- and Levantilide-Producers

Micromonospora sp. AKA109 is a producer of akazaoxime and A-76356, whereas Micromonospora sp. AKA38 is that of levantilide C. We aimed to clarify their taxonomic positions and identify biosynthetic gene clusters (BGCs) of these compounds. In 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequence analyses, strains AKA109 and AKA38 were the most closely related to Micromonospora humidisoli MMS20-R2-29T and Micromonospora schwarzwaldensis HKI0641T, respectively. Although Micromonospora sp. AKA109 was identified as M. humidisoli by the gyrB sequence similarity and DNA–DNA relatedness based on whole genome sequences, Micromonospora sp. AKA38 was classified to a new genomospecies. M. humidisoli AKA109 harbored six type-I polyketide synthase (PKS), one type-II PKS, one type-III PKS, three non-ribosomal peptide synthetase (NRPS) and three hybrid PKS/NRPS gene clusters, among which the BGC of akazaoxime and A-76356 was identified. These gene clusters are conserved in M. humidisoli MMS20-R2-29T. Micromonospora sp. AKA38 harbored two type-I PKS, one of which was responsible for levantilide C, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters. We predicted products derived from these gene clusters through bioinformatic analyses. Consequently, these two strains are revealed to be promising sources for diverse non-ribosomal peptide and polyketide compounds.


Introduction
Actinomycetes are Gram stain-positive and filamentous bacteria with high G + C contents in genomic DNAs. They are well known as a promising source for pharmacologically useful bioactive substances with diverse chemistries, from which many pharmaceuticals were developed and are clinically used [1]. The genus Streptomyces is the representative of actinomycetes, and its main habitat is soil. However, soil environments are extensively searched for novel actinomycetes, and consequently, it is getting harder to isolate novel actinomycetal strains from the same environments. In contrast, marine environments are attracting attention as rich sources of underexplored actinomycetes. Indeed, we have discovered new and diverse bioactive secondary metabolites from marine actinomycetes [2][3][4][5][6][7][8][9]. Micromonospora strains are frequently isolated from marine environments. Many bioactive substances are reported from this genus [10,11]. We previously isolated Micromonospora sp. AKA109 and Micromonospora sp. AKA38 from deep sea water. From Micromonospora sp. AKA109, a new compound named akazaoxime (1, Figure 1) was discovered, along with a known compound, A-76356 (2, Figure 1). Akazaoxime and A-76356 are enteromycin-class antibiotics. Incorporation experiments of labelled precursors suggested these two compounds are biosynthesized from glycine, leucin and propionate. Akazaoxime exhibits antibacterial activity to Gram-positive Kocuria rhizophila, whereas A-76356 is active against filamentous fungi such as the plant pathogen Glomerella cingulata [12]. Micromonospora sp. AKA38 produces levantilide C (3, Figure 1), which is a 20-membered from glycine, leucin and propionate. Akazaoxime exhibits antibacterial activity to Grampositive Kocuria rhizophila, whereas A-76356 is active against filamentous fungi such as the plant pathogen Glomerella cingulata [12]. Micromonospora sp. AKA38 produces levantilide C (3, Figure 1), which is a 20-membered macrolide and exhibits antiproliferative activities against several tumor cell lines [13]. Biosynthetic gene clusters (BGCs) of these compounds have not been identified yet, although identification of BGCs plays an important role in developments in combinatorial biosynthesis and synthetic biology. Polyketides such as macrolide backbones are biosynthesized by the assemblage of acyl-CoAs as building blocks. The assembly is catalyzed by polyketide synthases (PKSs). PKSs are classified by three types. Type-I PKSs are large modular enzymes composed of multiple catalytic domains. Polyketide chains are synthesized according to the co-linearity rule of assembly lines. Such a mechanism shows similarity to that in the biosynthesis of non-ribosomal peptides by non-ribosomal peptide synthetases (NRPSs), which is based on assembly of amino acids as building blocks. NRPSs as well as type-I PKSs are large and modular enzymes with multiple catalytic domains, and they accord to the co-linearity rule [14,15]. Polyketide chains for macrolide compounds are synthesized by type-I PKSs. Backbones synthesized by type-I PKSs and/or NRPSs can be predicted from their domain organizations by bioinformatic analysis [14,15]. In contrast, type-II PKSs are composed of three monofunctional enzymes, ketosynthase α (KSα), KSβ (chain length factor), and acyl carrier protein (ACP). Differently from type-I PKSs, these three enzymes iteratively catalyze multiple chain elongation steps. The main products of type-II PKSs are aromatic compounds [16]. Type-III PKSs are not multimodular or composed of abovementioned three enzymes, but stand alone with a KS domain and iteratively catalyze the assembly of the acyl-CoA unit [17]. Genome analyses revealed that half to three quarters of the secondary metabolite-BGCs in each actinomycetal genome are associated with PKSs or NRPSs. This suggests that polyketides, non-ribosomal peptides, and their hybrid compounds, which are derived from hybrid PKS/NRPS gene clusters, are main secondary metabolites in actinomycetes [18].
In the present study, we classified Micromonospora sp. AKA109 and Micromonospora sp. AKA38 at species level. Next, we identified BGCs for akazaoxime/A-76356 and levantilide C through analysis of PKS and NRPS gene clusters in their genomes. The analysis revealed the potential of the two strains to act as producers of diverse polyketideand nonribosomal peptide-compounds. These results are useful to elucidate potential products of each strain. Polyketides such as macrolide backbones are biosynthesized by the assemblage of acyl-CoAs as building blocks. The assembly is catalyzed by polyketide synthases (PKSs). PKSs are classified by three types. Type-I PKSs are large modular enzymes composed of multiple catalytic domains. Polyketide chains are synthesized according to the co-linearity rule of assembly lines. Such a mechanism shows similarity to that in the biosynthesis of non-ribosomal peptides by non-ribosomal peptide synthetases (NRPSs), which is based on assembly of amino acids as building blocks. NRPSs as well as type-I PKSs are large and modular enzymes with multiple catalytic domains, and they accord to the co-linearity rule [14,15]. Polyketide chains for macrolide compounds are synthesized by type-I PKSs. Backbones synthesized by type-I PKSs and/or NRPSs can be predicted from their domain organizations by bioinformatic analysis [14,15]. In contrast, type-II PKSs are composed of three monofunctional enzymes, ketosynthase α (KSα), KSβ (chain length factor), and acyl carrier protein (ACP). Differently from type-I PKSs, these three enzymes iteratively catalyze multiple chain elongation steps. The main products of type-II PKSs are aromatic compounds [16]. Type-III PKSs are not multimodular or composed of abovementioned three enzymes, but stand alone with a KS domain and iteratively catalyze the assembly of the acyl-CoA unit [17]. Genome analyses revealed that half to three quarters of the secondary metabolite-BGCs in each actinomycetal genome are associated with PKSs or NRPSs. This suggests that polyketides, non-ribosomal peptides, and their hybrid compounds, which are derived from hybrid PKS/NRPS gene clusters, are main secondary metabolites in actinomycetes [18].
In the present study, we classified Micromonospora sp. AKA109 and Micromonospora sp. AKA38 at species level. Next, we identified BGCs for akazaoxime/A-76356 and levantilide C through analysis of PKS and NRPS gene clusters in their genomes. The analysis revealed the potential of the two strains to act as producers of diverse polyketide-and nonribosomal peptide-compounds. These results are useful to elucidate potential products of each strain.

Materials and Methods
Micromonospora strains AKA109 and AKA38 were isolated from deep sea water collected in Shizuoka, Japan, maintained as TP-A0907 and TP-A0908, respectively, in Toyama Prefectural University, and have been deposited to and are available from the NBRC culture collection as NBRC 113680 and NBRC 113681, respectively. The 16S rRNA genes were Life 2023, 13, 542 3 of 16 amplified by PCR using 9F and 1541R primers. The amplicons were sequenced by the method described in our previous report [19]. Type strains showing high 16S rRNA gene sequence similarities to AKA109 and AKA38 were searched using the EzBioCloud web server [20]. Phylogenetic trees based on 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequences were reconstructed by the neighbor-joining method using ClustalX 2.1. Whole genomes were sequenced using PacBio, as reported [21]. Draft genome sequences of strains AKA109 and AKA38 were deposited to DDBJ under the accession numbers of BNEH01000001-BNEH01000007 and BNEI01000001-BNEI01000011, respectively. A phylogenomic tree was reconstructed using the TYSG server [22]. DNA-DNA relatedness was calculated by digital DNA-DNA hybridization (DDH) using the Genome-to-Genome Distance Calculator 2.1 (GGDC) [23], and DDH estimates by the Formula 2 were employed. PKS and NRPS gene clusters in the whole genome were searched, and their domains were determined using antiSMASH [24]. The products were predicted by reviewing module numbers and domain organizations in PKSs and NRPSs, the substrates of acyltransferase (AT) and adenylation (A) domains, and orthologs searched by BLAST, in addition to results of ClusterBlast in antiSMASH.

Materials and Methods
Micromonospora strains AKA109 and AKA38 were isolated from deep sea water collected in Shizuoka, Japan, maintained as TP-A0907 and TP-A0908, respectively, in Toyama Prefectural University, and have been deposited to and are available from the NBRC culture collection as NBRC 113680 and NBRC 113681, respectively. The 16S rRNA genes were amplified by PCR using 9F and 1541R primers. The amplicons were sequenced by the method described in our previous report [19]. Type strains showing high 16S rRNA gene sequence similarities to AKA109 and AKA38 were searched using the EzBioCloud web server [20]. Phylogenetic trees based on 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequences were reconstructed by the neighbor-joining method using ClustalX 2.1. Whole genomes were sequenced using PacBio, as reported [21]. Draft genome sequences of strains AKA109 and AKA38 were deposited to DDBJ under the accession numbers of BNEH01000001-BNEH01000007 and BNEI01000001-BNEI01000011, respectively. A phylogenomic tree was reconstructed using the TYSG server [22]. DNA-DNA relatedness was calculated by digital DNA-DNA hybridization (DDH) using the Genome-to-Genome Distance Calculator 2.1 (GGDC) [23], and DDH estimates by the Formula 2 were employed. PKS and NRPS gene clusters in the whole genome were searched, and their domains were determined using antiSMASH [24]. The products were predicted by reviewing module numbers and domain organizations in PKSs and NRPSs, the substrates of acyltransferase (AT) and adenylation (A) domains, and orthologs searched by BLAST, in addition to results of ClusterBlast in antiSMASH.

Classification of Micromonospora Strains AKA109 and AKA38
In the 16S rRNA gene sequence analysis, Micromonospora sp. AKA109 showed 100% similarity to Micromonospora humidisoli MMS20-R2-29 T , whereas Micromonospora sp. AKA38 showed 99.9% similarity to Micromonospora schwarzwaldensis HKI0641 T as the closest. In the phylogenetic tree shown in Figure 2, strain AKA109 formed an independent clade with M. humidisoli MMS20-R2-29 T , whereas strain AKA38 did that with M. schwarzwaldensis HKI0641 T .  We next reconstructed a phylogenetic tree based on gyrB sequences, as shown in Figure 3, since gyrB sequences are recognized to be more suitable than 16S rRNA gene sequences for phylogenetic classification and identification [25]. In this tree, M. humidisoli and M. schwarzwaldensis were also phylogenetically the closest species of strains AKA109 and AKA38, respectively. The gyrB sequence similarity between Micromonospora sp. AKA109 and M. humidisoli MMS20-R2-29 T was 99.0%. Since 98.5% in gyrB sequence similarity is recognized to correspond to 70% in DNA-DNA relatedness [25,26], Micromonospora sp. AKA109 is likely M. humidisoli. In contrast, the gyrB sequence similarity between Micromonospora sp. AKA38 and M. schwarzwaldensis HKI0641 T was 97.4%, which is much below than 98.5%; therefore, Micromonospora sp. AKA38 is considered an independent new genomospecies. sequences for phylogenetic classification and identification [25]. In this tree, M. humidi and M. schwarzwaldensis were also phylogenetically the closest species of strains AKA and AKA38, respectively. The gyrB sequence similarity between Micromonospora AKA109 and M. humidisoli MMS20-R2-29 T was 99.0%. Since 98.5% in gyrB seque similarity is recognized to correspond to 70% in DNA-DNA relatedness [25, Micromonospora sp. AKA109 is likely M. humidisoli. In contrast, the gyrB seque similarity between Micromonospora sp. AKA38 and M. schwarzwaldensis HKI0641 T w 97.4%, which is much below than 98.5%; therefore, Micromonospora sp. AKA38 considered an independent new genomospecies. Additionally, a phylogenomic tree was reconstructed with type strains whose wh genome sequences are published ( Figure 4). The phylogenetic relationships w correlated to those in phylogenetic trees of Figures 1 and 2. DNA-DNA relatedn estimated by digital DDH, between Micromonospora sp. AKA109 and M. humidi MMS20-R2-29 T was 93.5%. As this value is much higher than 70%, which is the establish cut-off for species delineation [27][28][29], strain AKA109 was identified to be M. humidis In contrast, DNA-DNA relatedness between Micromonospora sp. AKA38 and the ot strains shown in Figure 4 were less than 41.4%. This result also shows Micromonospora AKA38 to be an independent genomospecies.  Additionally, a phylogenomic tree was reconstructed with type strains whose whole genome sequences are published ( Figure 4). The phylogenetic relationships well correlated to those in phylogenetic trees of Figures 1 and 2. DNA-DNA relatedness, estimated by digital DDH, between Micromonospora sp. AKA109 and M. humidisoli MMS20-R2-29 T was 93.5%. As this value is much higher than 70%, which is the established cut-off for species delineation [27][28][29], strain AKA109 was identified to be M. humidisoli. In contrast, DNA-DNA relatedness between Micromonospora sp. AKA38 and the other strains shown in Figure 4 were less than 41.4%. This result also shows Micromonospora sp. AKA38 to be an independent genomospecies.

PKS and NRPS Gene Clusters in the Whole Genome of M. humidisoli AKA109
Six type-I PKS, one type-II PKS, one type-III PKS, three NRPS and three hybrid PKS/NRPS gene clusters were encoded in the genome of Micromonospora sp. AKA109. Type-I PKS gene cluster 1 (t1pks-1) encoded three PKSs, whose domain organization was almost identical to those (KS AT m ACP KS AT m DH KR ACP KS AT m DH KR ACP, KS AT m DH KR ACP KS AT m DH KR ACP, KS AT m / mm/em DH KR ACP TD) of camporidine-, argimycin-and streptazone-BGCs [30][31][32]. However, t1pks-1 lacked the KR domain (underlined in the previous brackets) present in CamD, ArpII and StzC. Although the substrate of the last AT domain in t1pks-1 was methylmalonyl-CoA, those in ArpIII and StzB are malonyl-CoA. Thus, product(s) of t1pks-1 may resemble camporidine, argimycin or streptazone, but will be different from these. PKSs encoded in t1pks-2, t1pks-3 and t1pks-4 did not show high sequence similarities to PKSs whose products have been identified. Thus, the products of these PKS gene clusters were not predicted. The domain organization, KS/AT/KR/DH, of the PKS encoded by TPA0907_18690 in t1pks-3 is well known as that of iterative PKSs for enediyne syntheses. Hence, the products of t1pks-3 may include an enediyne moiety. T1pks-5 encoded five PKSs. These PKSs showed high similarities to those in the marinolactam-BGC (mrl) [33]. Their domain organization was identical to that of mrl except for the presence of a DH domain in the first module of MrlB, which is absent in that of TPA0907_35890. Therefore, we annotated this cluster to be responsible for a marinolactum congener. As genes in t1pks-6 showed high similarities to those in the amycomicin-BGC, the product was predicted to be amycomicin. Products of type-II PKS gene cluster 1 (t2pks-1) were predicted to be an aromatic compound. Type-III PKS gene cluster 1 (t3pks-1) showed similarity to agq, which is the BGC of alkyl-O-dihydrogeranyl-methoxyhydroquinone [34]. Three NRPS gene clusters (nrps-1, nrps-2, and nrps-3) did not show high similarities to those whose products are elucidated, suggesting that they are orphan gene clusters. Although the product of nrps-2 was unpredictable because its NRPS was not multimodular, those of nrps-1 and nrps-3 were predicted as dipeptide and tetrapeptide, respectively, as shown in Table 1. Hybrid PKS/NRPS gene clusters 1 and 2 (pks/nrps-1 and pks/nrps-2) were orphan. The domain organization of pks/nrps-1 was unusual, because thioesterase (TE) domain is not present at the terminal, but as the first domain. Hence, it is doubtful that the cluster works to synthesize hybrid polyketide/non-ribosomal peptide compounds. The product derived from pks/nrps-2 was predicted to be a hybrid polyketide/non-ribosomal peptide compound including Asn and Ser residues.  We considered pks/nrps-3 to be the BGC for akazaoxime and A-76356, according to its domain organization and the biosynthetic pathway revealed by incorporation of labeled precursors [12]. These two compounds have been reported to be synthesized from glycine, leucine, and propionate. Similarly, pks/nrps-3 encodes two NRPS and one PKS, which incorporate two amino acids and one acyl-CoA, respectively, to the product. One of the amino acids was predicted to be leucine, although the other was bioinformatically not. Presence of a KR domain in the PKS well accounts for hydration of the keto group derived from carboxyl group of leucine. The cluster encoded a diiron oxygenase and a nitronate O-methyltransferase, which are essential to form aldoxime functionality and an O-methyl nitronic acid moiety [35]. We predicted the biosynthetic pathway of akazaoxime and A-76356, as shown Figure 5. A glycine molecule is loaded on the NRPS encoded by TPA0907_56660. Its amino group is converted to an aldoxime functionality through an intermediate by the diiron oxygenase, as reported in the biosynthesis of althiomycin [35,36]. If the methyltransferase encoded by TPA0907_56720 acts the intermediate, the amino group is converted to O-methyl nitronic acid moiety, as reported in the biosynthesis of enteromycin carboxamide [35]. To the modified glycine molecules, leucine and methylmalonyl-CoA are bound by the other NRPS (TPA0907_56840) and the PKS (TPA0907_56670). Finally, the chains are released from the PKS to yield akazaoxime (1) and A-76356 (2), respectively. incorporated by A domains are indicated as 3-letter abbreviations in subscript just after A.
We considered pks/nrps-3 to be the BGC for akazaoxime and A-76356, according to its domain organization and the biosynthetic pathway revealed by incorporation of labeled precursors [12]. These two compounds have been reported to be synthesized from glycine, leucine, and propionate. Similarly, pks/nrps-3 encodes two NRPS and one PKS, which incorporate two amino acids and one acyl-CoA, respectively, to the product. One of the amino acids was predicted to be leucine, although the other was bioinformatically not. Presence of a KR domain in the PKS well accounts for hydration of the keto group derived from carboxyl group of leucine. The cluster encoded a diiron oxygenase and a nitronate O-methyltransferase, which are essential to form aldoxime functionality and an O-methyl nitronic acid moiety [35]. We predicted the biosynthetic pathway of akazaoxime and A-76356, as shown Figure 5. A glycine molecule is loaded on the NRPS encoded by TPA0907_56660. Its amino group is converted to an aldoxime functionality through an intermediate by the diiron oxygenase, as reported in the biosynthesis of althiomycin [35,36]. If the methyltransferase encoded by TPA0907_56720 acts the intermediate, the amino group is converted to O-methyl nitronic acid moiety, as reported in the biosynthesis of enteromycin carboxamide [35]. To the modified glycine molecules, leucine and methylmalonyl-CoA are bound by the other NRPS (TPA0907_56840) and the PKS (TPA0907_56670). Finally, the chains are released from the PKS to yield akazaoxime (1) and A-76356 (2), respectively.

PKS and NRPS Gene Clusters in the Whole Genome of Micromonospora sp. AKA38
Micromonospora sp. AKA38 harbored two type-I PKS, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters in its genome, as listed in Table  2.

PKS and NRPS Gene Clusters in the Whole Genome of Micromonospora sp. AKA38
Micromonospora sp. AKA38 harbored two type-I PKS, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters in its genome, as listed in Table 2.
T1pks-8 is a large type-I PKS gene cluster encoding 13 PKSs, which form 33 modules. The product was predicted to be quinolidomicin based on the domain organization and similarities to quinolidomicin's PKSs (QmnA1 to QmnA13) [37]. The gene cluster is widely distributed in the genus Micromonospora [38]. The product of t2pks-2 could not be predicted because the type-II PKSs did not show high sequence similarities to enzymes for the reported compounds. In most type-II PKS gene clusters, an ACP is encoded downstream of KSβ (CLF), but the ACP of t2pks-2 is upstream of KSα and includes a cyclase domain. Two gene clusters, t3pks-1 and pks/nrps-2, asterisked in the tables, were orthologs of those present in M. humidisoli AKA109. The other gene clusters, such as nrps-4, nrps-5, pks/nrps-4, pks/nrps-5, pks/nrps-6 and pks/nrps-7, were orphan, and their products were predicted as shown in Table 2. In pks/nrps-7, two type-I PKSs whose domain organizations are KS-AT-KR-DH and KS-AT-ACP, respectively and one type-III PKS were encoded in addition to NRPSs. The domain pair, KR-DH, observed in one of the type-I PKSs is known to be specific for PksE. Therefore, the product of pks/nrps-7 will include an enediyne moiety [39].  We annotated t1pks-7 as the BGC of levantilide C, according to its domain organization and the chemical structure. The cluster encoded three PKSs including a loading module and eleven modules to incorporate acyl-CoAs in the polyketide chain, as shown in Figure 6. The chemical structure predicted by the domain organization well matched to that of levantilide C. DH and ER domains in module 3 and the DH domain in module 8 would be inactive considering the actual chemical structure of levantilide C. A hydroxyl group is present at C-10 in levantilide C, and it does not form by polyketide biosynthesis. Because a cytochrome P450 is encoded near the PKSs in the gene cluster as TPA0908_40790, the hydroxyl group is likely introduced by the cytochrome P450. We annotated t1pks-7 as the BGC of levantilide C, according to its domain organization and the chemical structure. The cluster encoded three PKSs including a loading module and eleven modules to incorporate acyl-CoAs in the polyketide chain, as shown in Figure 6. The chemical structure predicted by the domain organization well matched to that of levantilide C. DH and ER domains in module 3 and the DH domain in module 8 would be inactive considering the actual chemical structure of levantilide C. A hydroxyl group is present at C-10 in levantilide C, and it does not form by polyketide biosynthesis. Because a cytochrome P450 is encoded near the PKSs in the gene cluster as TPA0908_40790, the hydroxyl group is likely introduced by the cytochrome P450.  Table 1. dh, inactive DH; er, inactive ER.
T1pks-8 is a large type-I PKS gene cluster encoding 13 PKSs, which form 33 modules. The product was predicted to be quinolidomicin based on the domain organization and similarities to quinolidomicin's PKSs (QmnA1 to QmnA13) [37]. The gene cluster is  Table 1. dh, inactive DH; er, inactive ER.

Specificity of the PKS and NRPS Gene Clusters in Each Strain
We conducted a BLAST search to investigate whether the gene clusters identified in this study are specific in each strain or present in the other strains. All the PKSs and NRPSs of M. humidisoli AKA109 were also present in M. humidisoli MMS20-R2-29 T ( Table 3). As the TPA0907_16820 homolog in M. humidisoli MMS20-R2-29 T is not well sequenced, it was not hit in the search. Although a homolog of TPA0907_20190 was also present in M. humidisoli MMS20-R2-29 T , it is not described in the table because its sequence identity/similarity were lower (99/98 in%) than those of Micromonospora sp. RL09-050-HVF-A.  Among eleven gene clusters of Micromonospora sp. AKA38, seven (t1pks-8, t2pks-2, t3pks-1, pks/nrps-2, pks/nrps-4, pks/nrps-5 and pks/nrps-7) were present in other strains with high sequence identity/similarity, although TPA0908_54560, TPA0908_54550 and TPA0908_54470 in pks/nrps-7 were not observed, suggesting pks/nrps-7 orthologs in other strains may be partial or not completely sequenced. Except for pks/nrps-4, the closest genes were present in Micromonospora sp. RP3T and their identity/similarity values were quite high. In contrast, four gene clusters, t1pks-7, nrps-4, nrps-5 and pks/nrps-6, were not present in other strains because their BLAST top hits showed low identity/similarity values. This suggests that they are novel and specific to strain AKA38.

Discussion
Many strains found as producers of new bioactive substances have not been classified yet at species level. Consequently, relationships between products and taxonomic positions of the producer are not well understood. In this study, we classified Micromonospora sp. AKA109, a producer of akazaoxime and A-76356, to M. humidisoli [40]. In contrast, Micromonospora sp. AKA38, a producer of levantilide C, was revealed to be a novel genomospecies. If Micromonospora sp. AKA38 is characterized in detail [41], it can be proposed as a new Micromonospora species because it was not classified to known species. M. humidisoli is very recently proposed, and its type strain, MMS20-R2-29 T , was isolated from riverside soil. It is explained that its growth occurs in the presence of 0-2% NaCl, with optimal growth at 0% NaCl [40]. In contrast, strain AKA109 was isolated from deep sea water with a higher Life 2023, 13, 542 13 of 16 salt concentration. To the best of our knowledge, this is the first report on marine-derived M. humidisoli.
Recently, genome mining has often been used when searching for new compounds. However, if researchers find an unknown BGC that appears novel by genome mining, it may be a BGC for known compounds, because many BGCs of known compounds have not been identified, and consequently, they are considered BGCs for new compounds. Thus, BGCs of known compounds need to be identified for more effective genome mining if the BGCs have not been unidentified. We here identified the BGC of akazaoxime and A-76356, and that of levantilide C from Micromonospora sp. AKA109 and Micromonospora sp. AKA38, respectively. This is the first report on the BGCs and biosynthetic pathways of these compounds.
Micromonospora sp. AKA109, classified to M. humidisoli, harbored fourteen PKS and NRPS gene clusters, all of which are also present in M. humidisoli MMS20-R2-29 T . This well supports our idea that members of the same species possess similar sets of PKS and NRPS gene clusters [42][43][44]. Micromonospora sp. AKA38, classified as a new genomospecies, harbored eleven PKS and NRPS gene clusters. Although seven of them were present in other strains, such as Micromonospora sp. RP3T and Micromonospora sp. WMMA2032, the remaining four are not found in any other strains. If a strain is taxonomically novel at the species level, it may possess new PKS and/or NRPS gene clusters.
Although PKS and NRPS gene clusters found from our two strains include BGCs of known compounds such as amycomicin, alkyl-O-dihydrogeranyl-methoxyhydroquinone and quinolidomicin, and congeners of known compounds, they include many orphan and unknown clusters. Their products were predicted to be novel at present. Thus, these two strains are expected to produce new and diverse polyketide and non-ribosomal peptide compounds.
Except for PKS and NRPS gene clusters, eleven putative secondary metabolite-biosynthetic gene clusters are present in each genome of M. humidisoli AKA109 and Micromonospora sp. AKA38 (Tables S1 and S2). The products, except for SapB, desferrioxamine, Nacetylglutaminylglutamine amide (NAGGN) and class II lanthipeptides of Micromonospora sp. AKA38, could not be predicted because there is less information on these types of gene clusters. SapB, desferrioxamine, NAGGN, three terpene, and one hybrid oligosaccharide/terpene gene cluster are conserved in the two strains. SapB, desferrioxamine and NAGGN are known as common secondary metabolites in actinomycetes. The numbers of gene clusters shown in Tables S1 and S2 did not exceed those of the PKS and NRPS gene clusters (Tables 1 and 2). This supports the assertion that polyketides and non-ribosomal peptides are major and diverse secondary metabolites, as previously reported [18].

Conclusions
We sequenced whole genomes of an akazaoxime-and A-76356-producer, Micromonospora sp. AKA109, and a levantilide C-producer, Micromonospora sp. AKA38. Micromonospora sp. AKA109 was identified as M. humidisoli, whereas Micromonospora sp. AKA38 was revealed to be a new genomospecies. Akazaoxime-and A-76356-BGC and levantilide C-one were identified from whole genome sequences of these two strains, respectively. M. humidisoli AKA109 harbored fourteen PKS and NRPS gene clusters, all of which were conserved in the type strain of M. humidisoli. Micromonospora sp. AKA38 harbored eleven PKS and NRPS gene clusters. Our bioinformatic analysis suggested their potential to synthesis diverse non-ribosomal peptides and polyketides.
Author Contributions: Conceptualization, H.K. and Y.I., methodology, T.T., resources, Y.I., data curation, H.K., writing-original draft preparation, H.K., writing-review and editing, Y.I., project administration, T.T. and Y.I., funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by a commissioned project from the Japan Patent Office.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The whole genome shotgun project of Micromonospora sp. AKA109 and Micromonospora sp. AKA38 have been deposited at GenBank under the accession numbers BNEH00000000 and BNEI00000000, respectively. BioProject accession numbers are PRJDB9818 and PRJDB9819. BioSample accession numbers are SAMD00228008 and SAMD00228009.